Defects in the mitochondrion, the energy-producing unit of cell, lead to a wide range of neurodegenerative disorders caused by decreased energy production, free radical damage, and perturbation to apoptotic pathways. The TIM22 import pathway in the mitochondrion mediates the import and assembly of inner membrane proteins. Components of this pathway include the soluble Tim8p-Tim13p and Tim9p-Timl0p complexes in the intermembrane space and the TIM22 (Tim12p, Tim18p, Tim22p, and Tim54p) complex at the inner membrane. Mutations in the Tim8p homolog in humans, DDP1 (deafness/dystonia protein), cause Mohr-Tranebjaerg syndrome (MTS) or deafness/dystonia syndrome. MTS symptoms begin with deafness, followed by blindness, dystonia, and mental deterioration later in life. Unlike many mitochondrial diseases that affect both muscular and neural tissue, MTS defects are specific to neural tissue. The goal of this proposal is to determine the underlying mechanisms of mitochondrial assembly in mammalian systems with the long-term goal of understanding the tissue specific defects in MTS. The specific aims are to develop mouse and single cell models for defects in mitochondrial protein import, to characterize the protein import pathway through heterologous expression in S. cerevisiae, and to identify and characterize substrates of the TIM22 protein import pathway. The proposed project will expand fundamental knowledge about the mechanism of protein import into mammalian cells. Also, these studies will develop new animal models that will potentially play a role in understanding the molecular basis of other mitochondrial diseases such as mitochondrial myopathies and neuropathies.